510 duo microscope

Cross sections of TA muscles were used to measure centrally nucleated fibers (CNFs) and minimal Feret's diameter (Briguet et al., 2004 (link)). Sections were stained as above but with rabbit anti-dystrophin (PAS-16734; Thermo Fisher Scientific), mounted in Vectashield ​+ ​DAPI as above and imaged by confocal microscopy with a Zeiss 510 Duo microscope (Carl Zeiss). DAPI labeling was evaluated with ImageJ (NIH, Bethesda, MD), for determination of centrally nucleated fibers. Measurements of minimal Feret's diameter were obtained with Zeiss LSM Image Browser (Carl Zeiss). A total of 312 myofibers from 5 Crym tg mice and 722 fibers from 5 control mice were analyzed.

FDB muscles were harvested bilaterally and digested in Dulbecco's modified Eagle's medium with 4 mg/mL type II collagenase (Gibco, Thermo Fisher Scientific, Waltham, MA) for 3 h at 37 °C. Tissue was transferred to FDB medium (Dulbecco's modified Eagle's medium with 2% BSA, 1 μL/mL gentamicin, and 1 μL/mL fungizone). Single myofibers were mechanically separated by trituration and allowed to incubate overnight. Isolated fibers were plated down on coverslips coated with Geltrex (A1413201; Thermo Fisher Scientific) for 2 h. Coverslips were fixed in 2% paraformaldehyde at room temperature for 15 min. They were then permeabilized with 0.25% TritonX-100 in PBS for 10 min and stained with antibody to μ-crystallin (H00001428-M03; Abnova, Taiwan), diluted 1:100, followed by Alexa Fluor 488, goat anti-mouse secondary antibody (A11029; Alexa Molecular Probes, Invitrogen), diluted 1:200, with the M.O.M. kit, as described above. Each incubation was for 1 h at room temperature. Samples were imaged on a Zeiss 510 Duo microscope (Carl Zeiss, Thornwood, NY).